Fifteen minutes after the intravenous injection of overnight starved, anaesthetized rats with insulin, the concentration of fructose 2,6-biphosphate was increased more than 2-fold in the hearts of these animals. Insulin injection also caused a 2-3 fold increase in the Vmax of phosphofructokinase-2 with no detectable change in Km values. The effect persisted after precipitation of the enzyme with polyethylene glycol or after gel filtration through Sephadex G-25.